JP4266377B2 - Scavenger for substances having an anionic substituent - Google Patents

Abstract

A polymer carrier to which a prescribed zinc complex group is binding directly or through a spacer, having property to bond, under a certain condition, to an anionic substituent (a phosphate group, for example), showing low solubility to a solvent (preferably insolubility to a solvent) as a whole, and being capable of capturing, separating and purifying easily a substance having an anionic substituent (a phosphate group, for example).

Description

  The present invention is useful for separating and purifying a substance having an anionic substituent (for example, a phosphate group), a polymer carrier bound with a predetermined zinc complex group, an anionic substituent (for example, containing the polymer carrier) The present invention relates to a capturing agent for a substance having a phosphate group), a capturing device filled with the capturing agent, and a capturing method thereof.

  As a method for analyzing a substance having a phosphate group, for example, a phosphorylated biological substance, a method using an enzyme immunoassay (ELISA) or a method using a radioisotope is known.

  Enzyme immunization utilizes the principle that an antibody (or an antigen) specifically binds to a target substance. Therefore, there is a problem that it is necessary to purify and obtain a large amount of the target substance in order to produce an antibody specific to the target substance. Furthermore, since the production of antibodies uses an immune reaction of animals, there is a problem that it takes time to produce antibodies. In addition, since an antibody against a phosphorylation site in a molecular structure of several kDa (Dalton) or less cannot be produced, a phosphorylated biological material having such a molecular structure cannot be analyzed by enzyme immunoassay. There is a problem.

Further, a method using a radioactive isotope, using radioactive isotope ^{32 P.} Therefore, there is a problem that it takes time to manage the radiation in the laboratory and the waste liquid.

  As a method for lowering the phosphoric acid concentration in the waste water, JP-A-11-57695 discloses a method using a composite metal hydroxide. On the other hand, in the field of medicine, polymers used for the treatment of hyperphosphatemia include guanidino group-bonded polymers described in JP-T-8-506846. However, a polymer in which a composite metal hydroxide and a guanidino group are bonded has a weak binding force to a phosphate group. Therefore, in order to capture a certain amount of phosphoric acid, there is a problem that a complex metal hydroxide or a polymer to which a guanidino group is bound must be used as a large amount of phosphate group binding substrate.

  On the other hand, as a compound that strongly binds to a phosphate group, Journal of the American Chemical Society (USA), 1991, Vol. 113, No. 23, p. There are macrocyclic polyamine zinc complexes described in 8935-8941. However, the macrocyclic polyamine zinc complex is soluble in the solvent. Therefore, there is a problem that it is very difficult to separate and purify a substance having a phosphate group bonded to a macrocyclic polyamine zinc complex in a solution.

  Therefore, in order to separate and purify a phosphoric acid-containing substance having a phosphate group, the property of strongly binding to a phosphate group that is one of anionic substituents and separation and purification under certain conditions There is a demand for a capture agent that has the property of being easy and that is safe and inexpensive.

  An object of the present invention is to provide a safe and inexpensive capture agent that binds to an anionic substituent (for example, a phosphate group) under a certain condition and is easy to separate and purify a substance having the substituent, as well as this capture agent. An object of the present invention is to provide a simple filling device and a quick and easy method for capturing the same.

  As a result of intensive studies to solve the problems of the prior art, the inventors of the present invention bonded a specific zinc complex group to a polymer support, and the polymer support became an anionic substituent (for example, a phosphate group). Since it has a strong binding property and exhibits poor solvent solubility (preferably solvent insolubility) as a whole, separation and purification are extremely easy, that is, it can be a useful scavenger for a substance having the substituent. I found it. Furthermore, the capture device for the substance filled with the capture agent and a method for capturing the substance using the capture agent have also been established, and the present invention has been completed.

｛式中、Ｒは、相互に同一又は異なっていてもよく、水素原子；炭素数が１〜１６であるアルキル基；アシル基、アルコキシカルボニル基、アシルアルキル基、アルコキシカルボニルアルキル基、カルボキシアルキル基、カルバモイルアルキル基、シアノアルキル基、ヒドロキシアルキル基、アミノアルキル基又はハロアルキル基（ここで、これらの基のアルキル部分の炭素数は、１〜１６である）；カルボキシル基；カルバモイル基；シアノ基；ヒドロキシル基；アミノ基；或いはハロゲノ基である｝で示される亜鉛錯体基が直接又はスペーサーを介して結合したポリマー担体である。 That is, the present invention (1) has the general formula (1):

{In the formula, Rs may be the same or different from each other, hydrogen atom; alkyl group having 1 to 16 carbon atoms; acyl group, alkoxycarbonyl group, acylalkyl group, alkoxycarbonylalkyl group, carboxyalkyl group , A carbamoylalkyl group, a cyanoalkyl group, a hydroxyalkyl group, an aminoalkyl group or a haloalkyl group (wherein the alkyl moiety of these groups has 1 to 16 carbon atoms); a carboxyl group; a carbamoyl group; a cyano group; It is a polymer carrier in which a zinc complex group represented by a hydroxyl group; an amino group; or a halogeno group} is bonded directly or via a spacer.

で示される亜鉛錯体基が直接又はスペーサーを介して結合したポリマー担体を含有する、アニオン性置換基を有する物質の捕捉剤である。 Further, the present invention (2) is the polymer carrier of the above invention (1) or the general formula (2)

A capturing agent for a substance having an anionic substituent containing a polymer carrier to which a zinc complex group represented by the formula (1) is bonded directly or via a spacer.

  Furthermore, the present invention (3) is the scavenger of the invention (2), wherein the anionic substituent is a phosphate group.

  The present invention (4) is the capture agent of the invention (2) or (3), wherein the capture agent is in the form of beads.

  Furthermore, the present invention (5) is the capture agent of the invention (2) or (3), wherein the capture agent is in the form of a plate.

  Moreover, this invention (6) is a capture agent of the said invention (2) or (3) whose capture agent is a form of a fiber.

  Furthermore, the present invention (7) relates to a substance having an anionic substituent, which is filled with any one of the capturing agents of the inventions (2) to (4) or (6) and has a function of being filtered by a filter. It is a capture device.

  Further, the present invention (8) includes an anionic substituent comprising a step of capturing a substance having an anionic substituent by binding it to any one of the capturing agents of the inventions (2) to (6). This is a method for capturing a substance.

  Furthermore, the present invention (9) is the method according to the invention (8), further comprising a step of dissociating a substance having an anionic substituent from the capturing agent after the capturing step.

FIG. 1 is the first half of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) diagram using a capture agent containing a polymer carrier to which a zinc complex group is bound. FIG. 2 is the latter half of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) diagram using a capture agent containing a polymer carrier to which a zinc complex group is bound. FIG. 3 is the first half of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) diagram using a capture agent containing a polymer carrier to which a complex group not containing zinc is bound. FIG. 4 is the latter half of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) diagram using a capture agent containing a polymer carrier to which a complex group not containing zinc is bound. In these figures, 1 is bovine serum albumin, 2 is chicken ovalbumin, 3 is bovine β-casein, 4 is bovine α _S1 -casein non-phosphorylated, 5 is bovine α _S1 -casein, and 6 is tris-hydrochloric acid. First mixed solution of buffer solution (pH 7.0) and 0.5M sodium chloride aqueous solution, 7 second mixed solution of tris-hydrochloric acid buffer solution (pH 7.0) and 0.5M sodium chloride aqueous solution, 8 is tris -Acetate buffer (pH 7.0) 1st time, 9 is Tris-acetate buffer (pH 7.0) 2nd time, 10 is Tris-acetate buffer (pH 7.0) 3rd time, 11 is Tris-acetate Buffer solution (pH 7.0) 4th time, 12 is 10 mM phosphate buffer solution (pH 7.0), 13 is 15 mM phosphate buffer solution (pH 7.0), 14 is 20 mM phosphate buffer solution (pH 7.0). 0), 15 is 25 mM phosphate buffer (pH 7.0), 16 is 30 mM phosphate buffer (pH 7.0), 17 is 35 mM phosphate buffer (pH 7.0), 18 is 40 mM phosphate buffer (pH 7.0), and 19 is 45 mM. Phosphate buffer (pH 7.0), 20 is 50 mM phosphate buffer (pH 7.0), 21 is 60 mM phosphate buffer (pH 7.0), and 22 is 70 mM phosphate buffer (pH 7.). 0), 23 is 80 mM phosphate buffer (pH 7.0), 24 is 90 mM phosphate buffer (pH 7.0), 25 is 100 mM phosphate buffer (pH 7.0), and 26 is 200 mM phosphate. Buffer (pH 7.0), 27 is 300 mM phosphate buffer (pH 7.0), 28 is 400 mM phosphate buffer (pH 7.0), 29 is 500 mM phosphate buffer (pH 7.0) Shows the line.

  First, the polymer carrier to which the zinc complex group represented by the general formula (1) or the general formula (2) is bonded will be described. The “polymer carrier” is not particularly limited as long as it is a polymer capable of binding to the group and functions as a “carrier” or “support”. However, it is preferable that the polymer carrier has resistance to a solvent and a reagent used when it is used, and has physical strength necessary for filtration and washing. In addition, those that do not affect the capture of a substance having an anionic substituent are suitable. Specific examples are polystyrene, polyethylene, polypropylene, polyacetylene (polyin), polyvinyl chloride, polyvinyl ester, polyvinyl ether, polyacrylic acid ester, polyacrylic acid, polyacrylonitrile, polyacrylamide, polymethacrylic acid ester, polymethacrylic acid. , Polymethacrylonitrile, polymethacrylamide, polyether, polyacetal, polyester, polyethylene terephthalate (polyethylene terephthalate), polycarbonate, polyamide, nylon, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polysulfide, polysulfone, polysulfone Name amide, polymer alloy, cellulose, dextran, agarose, chitosan, silica, etc. It can be. Moreover, in order to ensure further physical strength, it may be a polymer carrier having a crosslinked (crosslinked) structure. Specific examples of the crosslinked (crosslinked) agent include divinylbenzene, epichlorohydrin, N , N′-methylenebisacrylamide, 4,4′-diphenylmethane diisocyanate, and the like.

  This polymer carrier is bonded to the zinc complex group represented by the general formula (1) or the general formula (2) directly or via a spacer. Here, the spacer is a polymer introduced for the purpose of accelerating the bond with the trapping substance to the zinc complex group and increasing the degree of swelling in the solvent by separating the zinc complex group from the polymer carrier. Are exemplified by polyethylene glycol, polyacrylamide, polyethylene, polyamide, polyester and the like. Specific examples of the bonding mode between the zinc complex group and the polymer carrier or spacer include covalent bonds such as a carbon-carbon bond, an ester bond, a carbonyl bond, an amide bond, an ether bond, a sulfide bond, an amino bond, and an imino bond. Can be mentioned.

や、Ｚｎ^２＋ _２−Ｎ，Ｎ，Ｎ’，Ｎ’−テトラキス［（２−ピリジル）メチル］−１，３−ジアミノ−２−プロポキシド基が結合したＡｒｇｏＧｅｌ（登録商標）：

や、Ｚｎ^２＋ _２−Ｎ，Ｎ，Ｎ’，Ｎ’−テトラキス［（２−ピリジル）メチル］−１，３−ジアミノ−２−プロポキシド基が結合したＴＳＫｇｅｌ（登録商標）：

や、Ｚｎ^２＋ _２−Ｎ，Ｎ，Ｎ’，Ｎ’−テトラキス［（２−ピリジル）メチル］−１，３−ジアミノ−２−プロポキシド基が結合したＳｅｐｈａｒｏｓｅ（商標）：

等を挙げることができる。 Specific examples of the polymer carrier to which the zinc complex group represented by the general formula (1) according to the present invention is bonded are illustrated as follows: Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl ] TOYOPEARL (registered trademark) to which a 1,3-diamino-2-propoxide group is bonded:

Or ArgoGel (registered trademark) to which a Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group is bonded:

Or TSKgel (registered trademark) to which a Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group is bonded:

Or Sepharose (trademark) to which a Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group is bonded:

Etc.

や、Ｚｎ^２＋−サイクレン基が結合したＡｒｇｏＧｅｌ（登録商標）：

や、Ｚｎ^２＋−サイクレン基を結合したＴｅｎｔａＧｅｌ（登録商標）：

等を挙げることができる。 A specific example of the polymer carrier to which the zinc complex group represented by the general formula (2) is bonded according to the present invention is exemplified by TOYOPEARL (registered trademark) having a Zn ²⁺ -cyclene group bonded thereto:

Or ArgoGel (registered trademark) to which a Zn ²⁺ -cyclene group is bonded:

Or TentaGel (registered trademark) bonded with a Zn ²⁺ -cyclene group:

Etc.

An anionic substituent is a substituent having a negative charge. To illustrate a specific example of an anion, a divalent phosphate monoester anion (—OPO ₃ ²⁻ ), a monovalent phosphate diester anion (( ^{_{-O) 2 PO 2 -),}} 2 -valent phosphonate anion ^{_(-PO 3} 2-), 1 monovalent phosphonate anion ^{_{((-) 2 PO 2 -}} ), 1 monovalent carbonate anion (--OCO ₂ ^- ) Monovalent carboxylate anion (—CO ₂ ^— ), monovalent sulfate anion (—OSO ₃ ^— ), monovalent sulfonate anion (—SO ₃ ^— ) and the like.

The substance having a phosphate group is a substance having a divalent phosphate monoester anion (—OPO ₃ ²⁻ ). Specific examples of the substance include phosphorylated amino acids and phosphorylated amino acids. Residues, proteins having phosphorylated amino acid residues (phosphorylated proteins), polypeptides having phosphorylated amino acid residues (phosphorylated polypeptides), oligopeptides having phosphorylated amino acid residues (phosphorus) Oxidized oligopeptides), deoxyribonucleic acid (DNA), phospholipids, phosphorylated saccharides, phosphate components in blood, phosphate components in waste water, and the like.

  The zinc complex group itself represented by general formula (1) or general formula (2) is usually soluble in relation to the solvent used. However, when this is combined with the polymer carrier, it becomes hardly soluble (preferably insoluble) in relation to the solvent. Therefore, the polymer carrier according to the present invention can capture a substance having an anionic substituent (for example, a phosphate group) in a solution under suitable conditions, and can be filtered and washed from a solvent. The substance can be separated and purified from the solution quickly and easily. Furthermore, it is possible to use a scavenger for the quantitative determination of the substance.

  Specific examples of uses in the capture agent used as the form of beads include column carriers such as metal-immobilized affinity chromatography (IMAC) columns, and substances having phosphate groups such as phosphorylated proteins or deoxyribonucleic acid (DNA). Examples thereof include a packing material for a column for purification or concentration, magnetic beads for separating or purifying a substance having a phosphate group, a preparation for capturing a phosphate component in blood used for the treatment of hyperphosphatemia, and the like.

  Specific examples of applications in the capture agent used as a plate form include purification of a sample substrate, protein, deoxyribonucleic acid (DNA), etc. used in a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF MS) Or the chip | tip etc. which perform a detection can be mentioned.

  Specific examples of the use of the scavenger used as the fiber form include separation membranes and hollow fiber membrane filters used for separating a substance having a phosphate group and a substance having no phosphate group. .

  The capturing device for a substance having an anionic substituent (for example, a phosphate group) according to the present invention has a function of filling a capturing agent containing the polymer carrier or a capturing agent in the form of beads or fibers, and filtering it with a filter. Have. Here, the filter refers to a solution in which these capture agents and a substance having the substituent bonded to the capture agent and a substance not having the substituent not bonded to the capture agent are dissolved, or these. Is a separating material introduced for the purpose of separating a solution in which a substance having a substituent dissociated from these scavengers and a substance having the substituent dissociated by simple individual liquid separation. Sintered metal filter, membrane filter, ultrafiltration membrane, asbestos, glass wool, polyethylene, polypropylene, polytetrafluoroethylene, polyethersulfone, cellulose and the like. Moreover, this filter may be above and below these scavengers, and has the effect of suppressing the flow range of these scavengers by sandwiching these scavengers between two filters.

When the specific example of the capture | acquisition device of the substance which has an anionic substituent (especially phosphate group) based on this invention is illustrated, Zn2 ⁺ _2- N, N, N ', N'-tetrakis [(2-pyridyl) methyl] A TOYOPEARL (registered trademark) column to which a 1,3-diamino-2-propoxide group is bonded, Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3- diamino-2-propoxide group bound Sepharose (TM) column, ^{Zn 2+} - TOYOPEARL the cyclen group bound (R) _{^{column, Zn 2+ 2 -N, N,}} N ', N'- tetrakis [(2- And a Sepharose (trademark) centrifugal filter unit to which a pyridyl) methyl] -1,3-diamino-2-propoxide group is bonded.

  The method for capturing a substance having an anionic substituent (for example, a phosphate group) of the present invention utilizes the principle that a zinc complex group bound to a polymer carrier binds to the substance. In addition, according to the scale or form at the time of capturing the substance, for example, by selecting a polymer carrier such as a bead, a plate, or a fiber, the substance can be captured according to the application.

  The method for capturing a substance having an anionic substituent (for example, a phosphate group) according to the present invention includes, for example, binding the substance and the polymer carrier according to the present invention under neutral conditions that are physiological conditions, and the like. For example, the polymer according to the present invention can be obtained by changing the pH of the solution, the acid having a buffering action and the salt thereof, the type of the salt and the salt, the concentration of the salt contained in the buffer solution or the solution, and the like. It is dissociated from the carrier. The substance can be captured and the captured substance can be released because the bond between the substance and the polymer carrier according to the present invention is, for example, the pH of the solution, an acid having a buffering action and its salt, or a base and its salt. This is because it varies depending on the type of the salt, the concentration of the salt contained in the buffer solution or the solution, and the like.

  Furthermore, the method for capturing a substance having an anionic substituent (for example, phosphate group) according to the present invention includes adding a chelating agent such as ethylenediaminetetraacetic acid (EDTA) to the bound substance in the present invention. Also included are those dissociated from such polymer carriers. The trapped substance can be released because, for example, a chelating agent such as ethylenediaminetetraacetic acid (EDTA) extracts the zinc ion from the bond between the substance and the polymer carrier according to the present invention. This is because the substance dissociates.

  Thus, separation and purification and recovery of a substance having an anionic substituent (for example, a phosphate group) using a rapid and easy capture method can be performed, for example, the pH of a solution, a buffering acid, its salt or base, and its It can be carried out freely by changing the kind of salt, the concentration of the salt contained in the buffer or the solution, or adding a chelating agent such as ethylenediaminetetraacetic acid (EDTA).

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples described below. In these examples, the amount of zinc ion (Zn ²⁺ ) supported (unit: mmol Zn ²⁺ / g or μmol Zn ²⁺ / mL) is a value representing the zinc ion content relative to the total amount of the polymer carrier to which the zinc complex group is bound. It is.

[Reference Example 1]
10 mL of TOYOPEARL (registered trademark) AF-Epoxy-650M, 6.9 g of cyclen, and 75 mL of ethanol solution of 2.2 g of potassium carbonate were stirred at 40 ° C. for 4 days. After the reaction solution was filtered and washed, 75 ml of an aqueous solution of the obtained beads and 12 g of zinc nitrate hexahydrate was stirred at 40 ° C. for 2 days. After filtering and washing the reaction solution, drying under reduced pressure yielded 12 g (0.3 mmol Zn ²⁺ / g) of TOYOPEARL (registered trademark) to which a Zn ²⁺ -cyclene group was bonded.

[Reference Example 2]
The columns with polyethylene filter, ^{Zn 2+} - TOYOPEARL the cyclen group bound ^{(R) 100mg (0.3mmol Zn 2+ / g} ) was filled with, ^{Zn 2+} - cyclen group bonded TOYOPEARL (TM ) A column was prepared.

[Reference Example 3]
A TOYOPEARL (registered trademark) column with a Zn ²⁺ -cyclene group bound thereto was swollen with 50 mM Tris-HCl buffer (pH 7.0). Add 1.0 mL of 10 mM sodium 4-nitrobenzoate solution adjusted with 50 mM Tris-HCl buffer (pH 7.0) to the column, and then add 2.0 mL of 50 mM Tris-HCl buffer (pH 7.0). And then spilled. The effluent was collected, and the capture rate of monovalent 4-nitrobenzoate anion from the effluent using an ultraviolet-visible spectrophotometer was 100%.

[Reference Example 4]
A TOYOPEARL (registered trademark) column with a Zn ²⁺ -cyclene group bound thereto was swollen with 50 mM Tris-HCl buffer (pH 7.0). 1.0 mL of 10 mM 4-nitrophenyl phosphate disodium solution adjusted with 50 mM Tris-HCl buffer (pH 7.0) was added to the column, and then 2.0 mL of 50 mM Tris-HCl buffer (pH 7.0). Was added and allowed to flow out. The effluent was collected, and the capture rate of divalent 4-nitrophenylphosphate anion from the effluent using an ultraviolet-visible spectrophotometer was measured to be 98%.

[Synthesis Example 5]
A methanol solution (500 mL) of 1,3-diamino-2-propanol (33 g), 2-pyridinecarboxaldehyde (116 g) and sodium cyanotrihydroborate (50 g) was stirred at room temperature for 3 days. After post-treatment, purification by column chromatography yielded 34 g of synthetic intermediate.

  225 mL of N, N-dimethylformamide solution of 18 g of synthetic intermediate, 12 g of methyl 6-bromomethylnicotinate and 14 g of potassium carbonate was stirred at 50 ° C. for 1 hour. After post-treatment, purification by column chromatography gave 22 g of methyl ester.

  Methanol ester solution 17g and 1.0M sodium hydroxide aqueous solution 83mL methanol solution 40mL were stirred at room temperature for 1 hour. After neutralization and purification by column chromatography, N- (5-carboxy-2-pyridyl) methyl-N, N ′, N′-tris [(2-pyridyl) methyl] -1,3-diamino-2 -13 g of propanol were obtained.

[Example 6]
TOYOPEARL (registered trademark) AF-Amino-650M 1.2 g and N- (5-carboxy-2-pyridyl) methyl-N, N ′, N′-tris [(2-pyridyl) methyl] -1,3-diamino A solution of 450 mg of 2-propanol, 210 mg of dicyclohexylcarbodiimide and 150 mL of 1-hydroxybenzotriazole in 10 mL of N, N-dimethylformamide was stirred at 40 ° C. for 18 hours. After filtering and washing the reaction solution, the obtained beads, 480 mg of zinc acetate dihydrate, and 7.0 mL of a 10 M aqueous sodium hydroxide solution (0.10 mL) were stirred at 40 ° C. for 18 hours. The reaction solution was filtered and washed, and then the obtained beads and 7.0 mL of a 1.0 M aqueous sodium perchlorate solution (3.0 mL) were stirred at 40 ° C. for 18 hours. When the reaction solution is filtered and washed and then dried under reduced pressure, the Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group is obtained. Bound TOYOPEARL® 1.0 g (0.5 mmol Zn ²⁺ / g) was obtained.

[Example 7]
TOYOPEARL in which Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group is bonded to a column with a polyethylene filter ( (Registered trademark) 100 mg (0.5 mmol Zn ²⁺ / g) was charged and Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2- A TOYOPEARL (registered trademark) column with a propoxide group attached thereto was prepared.

[Example 8]
A TOYOPEARL (registered trademark) column to which a Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group was bonded was treated with 50 mM Tris-HCl. Swelled with a buffer solution (pH 7.0) / acetonitrile (1/1). To the column was added 1.0 mL of 10 mM sodium 4-nitrobenzoate solution adjusted with 50 mM Tris-HCl buffer (pH 7.0) / acetonitrile (1/1) solution, and then 50 mM Tris-HCl buffer (pH 7). 0.0) / acetonitrile (1/1) solution was added and allowed to flow out. The effluent was collected, and the capture rate of monovalent 4-nitrobenzoate anion from the effluent using an ultraviolet-visible spectrophotometer was 100%. Subsequently, 50 mL of a 50 mM phosphate buffer (pH 3.0) / acetonitrile (1/1) solution was added and allowed to flow out. The effluent was collected, and the recovery rate of monovalent 4-nitrobenzoic acid anion from the effluent using an ultraviolet-visible spectrophotometer was 100%.

[Example 9]
A TOYOPEARL (registered trademark) column to which a Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group was bonded was treated with 50 mM Tris-HCl. Swelled with buffer (pH 7.0). 1.0 mL of 10 mM 4-nitrophenyl phosphate disodium solution adjusted with 50 mM Tris-HCl buffer (pH 7.0) was added to the column, and then 2.0 mL of 50 mM Tris-HCl buffer (pH 7.0). Was added and allowed to flow out. The effluent was collected, and the capture rate of divalent 4-nitrophenylphosphate anion was measured from the effluent using an ultraviolet-visible spectrophotometer. As a result, it was 100%. Subsequently, 50 mL of 50 mM phosphate buffer (pH 3.0) is added as an eluent to flow out, the effluent is collected, and divalent 4-nitrophenyl phosphate is collected from the effluent using an ultraviolet-visible spectrophotometer. The recovery rate of the anion was 89%, and 50 mL of 50 mM Tris-hydrochloric acid buffer (pH 7.0) was added as an eluent to flow out. The effluent was collected, and an ultraviolet-visible spectrophotometer Was used to measure the recovery of divalent 4-nitrophenylphosphate anion from the effluent, and it was 0%.

[Synthesis Example 10]
A methanol solution (500 mL) of 1,3-diamino-2-propanol (33 g), 2-pyridinecarboxaldehyde (116 g) and sodium cyanotrihydroborate (50 g) was stirred at room temperature for 3 days. After post-treatment, purification by column chromatography yielded 34 g of synthetic intermediate.

225 mL of N, N-dimethylformamide solution of 18 g of synthetic intermediate, 12 g of methyl 6-bromomethylnicotinate and 14 g of potassium carbonate was stirred at 50 ° C. for 1 hour. After post-treatment, purification by column chromatography gave 22 g of methyl ester.
A methanol solution (100 mL) of methyl ester compound (10 g) and ethylenediamine (23 g) was stirred at room temperature for 3 days. After working up and purifying by column chromatography, N- {5- [N- (2-amino) ethyl] carbamoyl-2-pyridyl} methyl-N, N ′, N′-tris [(2-pyridyl ) 10 g of methyl] -1,3-diamino-2-propanol was obtained.

[Synthesis Example 11]
NHS-activated Sepharose ™ 4FF 5.0 mL in 20 mM acetonitrile and N- {5- [N- (2-amino) ethyl] carbamoyl-2-pyridyl} methyl-N, N ′, N′-tris [( 2-Pyridyl) methyl] -1,3-diamino-2-propanol in 5.0 mL of 5.0 mM acetonitrile was stirred at 50 ° C. for 1 hour. The reaction solution was filtered and washed, and then washed with a 20 mM aqueous sodium carbonate solution to separate Sepharose to which N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propanol group was bonded. (Trademark) 5.0mL was obtained.

[Example 12]
Sepharose (trademark) 1.0 mL in which N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propanol group was bound to a column with a polypropylene filter was added. Filled and mixed solution of 100 mM 2- (N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.0) and 20 mM aqueous zinc acetate solution, 5.0 mL, 100 mM 2- (N-morpholino) ethanesulfonic acid (MES) Equilibrate with 5.0 mL of a mixed solution of buffer solution (pH 6.0) and 0.1 mM zinc acetate aqueous solution, and 5.0 mL of a mixed solution of Tris-hydrochloric acid buffer solution (pH 7.0) and 0.5 M sodium chloride aqueous solution. , Zn2 ⁺ _2- N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group bound thereto A rose ™ column was made.

[Example 13]
Bovine serum albumin (molecular weight) was added to a Sepharose ™ column to which a Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group was bound. 66,000 phosphorylated serine residue × 0) 50 μg, chicken ovalbumin (molecular weight 45,000 phosphorylated serine residue × 2) and bovine α _S1 -casein (molecular weight 24,000 phosphorylated serine residue × 8) 50 μg And 50 μg of bovine α _S1 -casein non-phosphorylated form (molecular weight 24,000 phosphorylated serine residue × 0) and bovine β-casein (molecular weight 25,000 phosphorylated serine residue × 5) were added. Subsequently, 1.0 mL of a mixed solution of Tris-hydrochloric acid buffer solution (pH 7.0) and 0.5 M aqueous sodium chloride solution was twice, and 1.0 mL of Tris-acetic acid buffer solution (pH 7.0) was 4 times, 10 mM. Phosphate buffer (pH 7.0) 1.0 mL, 15 mM phosphate buffer (pH 7.0) 1.0 mL, 20 mM phosphate buffer (pH 7.0) 1.0 mL, 25 mM phosphate buffer ( pH 7.0) 1.0 mL, 30 mM phosphate buffer (pH 7.0) 1.0 mL, 35 mM phosphate buffer (pH 7.0) 1.0 mL, 40 mM phosphate buffer (pH 7.0) 1.0 mL, 45 mM phosphate buffer (pH 7.0) 1.0 mL, 50 mM phosphate buffer (pH 7.0) 1.0 mL, 60 mM phosphate buffer (pH 7.0) 1.0 mL, 70 mM Phosphate buffer solution (pH 7.0) 1.0 mL, 80 1.0 mM mM phosphate buffer (pH 7.0), 1.0 mL 90 mM phosphate buffer (pH 7.0), 1.0 mL, 100 mM phosphate buffer (pH 7.0), 200 mM phosphate buffer (PH 7.0) 1.0 mL, 300 mM phosphate buffer (pH 7.0) 1.0 mL, 400 mM phosphate buffer (pH 7.0) 1.0 mL, 500 mM phosphate buffer (pH 7.0) ) Added 1.0 mL in order and let it flow out. The effluent was concentrated, spotted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then stained with Coomassie brilliant blue (CBB). The first half of the electropherogram is shown in FIG. 1, and the second half is shown in FIG. In FIGS. 1 and 2, bovine serum albumin having no phosphorylated serine residue first, followed by chicken ovalbumin, bovine β-casein, bovine α _S1 -casein in the order of decreasing number of phosphorylated serine residues. The outflow was confirmed.

[Example 14]
Sepharose in which a filter made of cellulose is packed in a micropipette tip and Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group is bound Sepharose (trademark) to which Zn2 ⁺ _2- N, N, N ', N'-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group was taken out from the (trademark) column Filled with 10 μL, then capped with cellulose filter, Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide A Sepharose ™ chip with attached groups was made.

[Example 15]
A micropipette with a Sepharose ™ chip attached with a Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group; 10 μL of a mixed solution of 0.22 mM p60 ^c-src peptide 521-533 and 0.18 mM phosphorylated p60 ^c-src peptide 521-533 prepared with 100 mM Tris-acetate buffer (pH 7.4) containing 0.50 M sodium nitrate was added. Inhaled and allowed to equilibrate for 5 minutes before exhaling. Then, it was washed 5 times with 10 μL of 100 mM Tris-acetate buffer (pH 7.4) containing 0.50 M sodium nitrate. Subsequently, the plate was washed 6 times with 10 μL of 10 mM phosphate buffer (pH 7.0), and the washings were collected and the purity was measured using high performance liquid chromatography. As a result, phosphorylated p60 ^c-src peptide 521-533 was obtained. Was 100% and p60 ^c-src peptide 521-533 was 0%.

[Example 16]
0.38M N- {5- [N- (2-amino) ethyl] carbamoyl-2-pyridyl} methyl prepared in a PS20 ProteinChip (registered trademark) Array with a 100 mM aqueous sodium bicarbonate solution / acetonitrile (1/3) solution -N, N ', N'-tris [(2-pyridyl) methyl] -1,3-diamino-2-propanol solution (3.0 μL) was placed and reacted at room temperature for 4 hours. Further, 3.0 μL of a 0.50M 2-aminoethanol solution prepared with a 100 mM sodium hydrogen carbonate aqueous solution was placed and reacted at room temperature for 4 hours. After washing, when washed with a 0.50 M zinc acetate solution adjusted with 100 mM 2- (N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.0), Zn ²⁺ ₂ -N, N, N ′, N A ProteinChip (registered trademark) Array to which a '-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group was bonded was obtained.

[Example 17]
To a ProteinChip (registered trademark) Array to which a Zn ²⁺ ₂ -N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propoxide group is bonded, 100 mM Tris-acetic acid A 0.94 mM 4-methylumbelliferyl disodium phosphate solution (3.0 μL) adjusted with a buffer solution (pH 7.4) was placed, equilibrated at room temperature for 1 hour, and then collected. The collected liquid was collected, and the capture rate of divalent 4-methylumbelliferyl phosphate anion was measured from the collected liquid using an ultraviolet-visible spectrophotometer. As a result, it was 16%.

[Comparative Example 1]
A column with a polyethylene filter was filled with 100 mg of TOYOPEARL (registered trademark) AF-Epoxy-650M and swollen with 50 mM Tris-HCl buffer (pH 7.0). 1.0 mL of 10 mM 4-nitrophenyl phosphate disodium solution adjusted with 50 mM Tris-HCl buffer (pH 7.0) was added to the column, and then 2.0 mL of 50 mM Tris-HCl buffer (pH 7.0). Was added and allowed to flow out. The effluent was collected, and the capture rate of divalent 4-nitrophenylphosphate anion from the effluent using an ultraviolet-visible spectrophotometer was measured and found to be 0.5%.

[Comparative Example 2]
Sepharose (trademark) 1.0 mL in which N, N, N ′, N′-tetrakis [(2-pyridyl) methyl] -1,3-diamino-2-propanol group was bound to a column with a polypropylene filter was added. The mixture was filled and equilibrated with 5.0 mL of a mixed solution of Tris-HCl buffer (pH 7.0) and 0.5 M aqueous sodium chloride solution. 50 μg of bovine serum albumin (molecular weight 66,000 phosphorylated serine residue × 0), 50 μg of chicken ovalbumin (molecular weight 45,000 phosphorylated serine residue × 2) and bovine α _S1 -casein (molecular weight 24,000 phosphorylated) Serine residue × 8) 50 μg and bovine α _S1 -casein non-phosphorylated type (molecular weight 24,000 phosphorylated serine residue × 0) and bovine β-casein (molecular weight 25,000 phosphorylated serine residue × 5) 50 μg Was added. Subsequently, 1.0 mL of a mixed solution of Tris-hydrochloric acid buffer solution (pH 7.0) and 0.5 M aqueous sodium chloride solution was twice, and 1.0 mL of Tris-acetic acid buffer solution (pH 7.0) was 4 times, 10 mM. Phosphate buffer (pH 7.0) 1.0 mL, 15 mM phosphate buffer (pH 7.0) 1.0 mL, 20 mM phosphate buffer (pH 7.0) 1.0 mL, 25 mM phosphate buffer ( pH 7.0) 1.0 mL, 30 mM phosphate buffer (pH 7.0) 1.0 mL, 35 mM phosphate buffer (pH 7.0) 1.0 mL, 40 mM phosphate buffer (pH 7.0) 1.0 mL, 45 mM phosphate buffer (pH 7.0) 1.0 mL, 50 mM phosphate buffer (pH 7.0) 1.0 mL, 60 mM phosphate buffer (pH 7.0) 1.0 mL, 70 mM Phosphate buffer solution (pH 7.0) 1.0 mL, 80 1.0 mM mM phosphate buffer (pH 7.0), 1.0 mL 90 mM phosphate buffer (pH 7.0), 1.0 mL, 100 mM phosphate buffer (pH 7.0), 200 mM phosphate buffer (PH 7.0) 1.0 mL, 300 mM phosphate buffer (pH 7.0) 1.0 mL, 400 mM phosphate buffer (pH 7.0) 1.0 mL, 500 mM phosphate buffer (pH 7.0) ) Added 1.0 mL in order and let it flow out. The effluent was concentrated, spotted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then stained with Coomassie brilliant blue (CBB). The first half of the electropherogram is shown in FIG. 3, and the second half is shown in FIG. 3 and 4, it was possible to confirm the outflow of bovine serum albumin, chicken ovalbumin, bovine β-casein, bovine α _S1 -casein from the beginning.

  The polymer carrier to which a predetermined zinc complex group according to the present invention is bonded binds to an anionic substituent (for example, a phosphate group) under a certain condition and is hardly soluble in a solvent (preferably solvent insoluble) as a whole. Therefore, it becomes a safe and inexpensive capturing agent that facilitates separation and purification of the substance having the substituent. Furthermore, the use of this polymer carrier provides a capture device that allows simple separation and purification with a filter, and a rapid and easy capture method for binding under neutral conditions and then dissociating under certain conditions.

FIG. 1 is the first half of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) diagram using a capture agent containing a polymer carrier to which a zinc complex group is bound. FIG. 2 is the latter half of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) diagram using a capture agent containing a polymer carrier to which a zinc complex group is bound. FIG. 3 is the first half of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) diagram using a capture agent containing a polymer carrier to which a complex group not containing zinc is bound. FIG. 4 is the latter half of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) diagram using a capture agent containing a polymer carrier to which a complex group not containing zinc is bound.

Claims (9)

General formula (1):

{In the formula, Rs may be the same or different from each other, hydrogen atom; alkyl group having 1 to 16 carbon atoms; acyl group, alkoxycarbonyl group, acylalkyl group, alkoxycarbonylalkyl group, carboxyalkyl group , A carbamoylalkyl group, a cyanoalkyl group, a hydroxyalkyl group, an aminoalkyl group or a haloalkyl group (wherein the alkyl moiety of these groups has 1 to 16 carbon atoms); a carboxyl group; a carbamoyl group; a cyano group; A polymer carrier to which a zinc complex group represented by hydroxyl group; amino group; or halogeno group is bonded directly or via a spacer. Containing the polymer responsible body ranging first claim of claim, scavenger substance having an anionic substituent.   The scavenger according to claim 2, wherein the anionic substituent is a phosphate group.   4. A capture agent according to claim 2 or claim 3, wherein the capture agent is in the form of beads.   4. A capture agent according to claim 2 or claim 3, wherein the capture agent is in the form of a plate.   4. A scavenger according to claim 2 or claim 3, wherein the scavenger is in the form of a fiber.   A capturing device for a substance having an anionic substituent, which is filled with the capturing agent according to any one of claims 2 to 4 or 6, and has a function of being filtered by a filter.   A method for capturing a substance having an anionic substituent, comprising a step of capturing a substance having an anionic substituent by binding it to the capturing agent according to any one of claims 2 to 6.   The method according to claim 8, further comprising the step of dissociating a substance having an anionic substituent from the capturing agent after the capturing step.

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